Influenza A virus infection results in ~100,000 hospitalizations and 36,000 deaths in the U.S. per year. Humans can be protected by vaccination against seasonal influenza strains. However, current influenza vaccines mostly induce antibodies to the virus'external glycoproteins. These antibodies neutralize the virus and prevent infection when given to naove mice. Because the glycoproteins are highly variable among influenza strains, neutralizing antibodies are primarily effective against the same viruses used in the vaccine. By contrast, vaccines against less variable viral proteins could protect against multiple strains. Such a vaccination strategy would facilitate preparation for worldwide (pandemic) spread of newly adapted viruses, such as H5N1 avian influenza. Immunizing mice with the highly conserved influenza A nucleoprotein (NP) elicits immunity against viruses of multiple serotypes, including H5N1 human isolates. Although NP-specific cytotoxic T cells might mediate this protection, a role for non-neutralizing antibody against NP cannot be excluded. Our preliminary results show a requirement for antibody in protection elicited by recombinant NP immunization. However, it is unknown how antibodies against an internal viral protein might function in protective immunity. The long-term objective of this study is to understand the protective potential of such non-neutralizing antibodies to influenza. This understanding could then be used to enhance cross-protection of human vaccines that could: 1) lessen the impact of seasonal flu, 2) prepare for unexpected strains and potentially pandemic outbreaks, and 3) provide long-term protection that would lessen the need for annual re-immunization, and thus have an economic benefit. We hypothesize that anti-NP antibodies promote immunity by binding to NP released from infected cells to form complexes that activate innate anti-viral mechanisms, and then enhance NP-specific T cell responses - ultimately leading to accelerated viral clearance. Therefore, SPECIFIC AIM 1 will determine what effector molecules (FcR, TLR, IFN12R, and complement) are required for NP-specific antibodies to protect naive mice. Because these molecules may promote viral clearance via T cell-mediated mechanisms, SPECIFIC AIM 2 will examine how anti-NP antibodies influence T cell responses and whether T cells are required for rNP-immune antibody-mediated protection. These aims will be addressed by immunizing mice with influenza NP and purifying the antibody generated in the serum. This NP-immune antibody will be transferred to mice deficient in antibody receptors, in other effector molecules, or in T cells. After infection of the recipients with influenza virus, morbidity (weight loss), and the levels of virus in the lung will be monitored. The results will be compared between recipients of immune and non-immune antibody, and between normal recipients and those deficient in the given factor being tested. Because the role of non-neutralizing antibody in influenza immunity is largely underappreciated, the results from these experiments will collectively demonstrate a novel understanding of the mechanisms for cross-protective immunity. PUBLIC HEALTH RELEVANCE Although current influenza vaccines can protect humans from seasonal outbreaks, they are limited to reacting to the same viruses used in the vaccine, but not to unexpected strains, such as avian H5N1 influenza. Our research will examine unique and unappreciated ways in which vaccination can induce immune reactions to a protein that is highly similar among all influenza A strains. Understanding these reactions could then enhance human vaccines to 1) react against multiple virus strains regardless of small (seasonal) or large (potentially pandemic) changes, and 2) provide long-term protection that would lessen the need for annual re- immunization.